Cardiac myocytes (CM, <50% of total cell number) and cardiac fibroblasts (CF, 40-60%) are the two major cell types in the myocardium that are highly interspersed, with one or more CF bordering each CM. Intercellular crosstalk is believed to play a central role in determining normal cardiac function and the cardiac remodeling response that ensues in response to many cardiovascular diseases, generally entails CM hypertrophy, CF activation and increased ECM production (fibrosis), and often leads to heart failure and arrhythmias. However, there is a fundamental gap in understanding of the functional importance and mechanisms of CM-CF communication, in part due to a lack of suitable experimental models. Since the intricate interspersion of CF and CM makes investigations of their crosstalk in situ extremely difficult, cell culture systems are required to investigate functional interactions between the two cell types in a direct and controllable manner. The objectives of this application are to advance in vitro co-culture approaches for CM and CF and to investigate the contributions of cell-cell interactions and paracrine effects as well as the functional significance of CF-to-CM crosstalk, both under physiological and pathophysiological conditions. Our long-term goal is to utilize the new experimental models to discover novel pathways that mediate and regulate the interaction between CM and CF in the heart and contribute to the remodeling response. The Specific Aims are (1) To develop micropatterned 2D CM-CF co-cultures with defined homo- and heterotypic interactions and to determine the effect of CF on CM morphology, gene expression and function; (2) To develop 3D CM-CF co-cultures with CF in interspersed or compact configuration and to determine the effect of CF on morphological and integrated functional responses of 3D microtissues. CM and CF size, phenotype, morphology and collagen production as well as CM functional changes (i.e., action potential propagation and calcium transients) will be examined. Molecular mechanisms will be investigated using cell-type-selected gene expression analysis via laser capture microdissection and electrophysiological studies. The proposed study is innovative because our multidisciplinary approach will lead to novel and complementary experimental models with enhanced control of CM-CF interactions (2D model) and with a distribution that mimics the ventricular myocardium (3D model). The models will enable investigations of the communication of cell types that are intricately linked in a tissue context in situ. The proposed research is significant because the models can serve as new platforms for unbiased molecular discovery of novel molecular pathways that mediate and regulate the interaction between CM and CF. the new pathways could serve as targets for the development of new strategies to counteract or prevent cardiac remodeling in response to hemodynamic stress. Furthermore, we anticipate that the models that will be developed and the insights gained in this project will facilitate research on intercellular crosstalk between other cell types in the cardiac field and beyond.